First molecular detection of Entamoeba gingivalis subtypes in individuals from Turkey

Abstract Entamoeba gingivalis is a parasitic protozoan that colonizes the human oral cavity and there are two subtypes (ST1 and ST2) that have been identified to date. However, there are no reports on the molecular detection or characterization of E. gingivalis in Turkey. The objective of this study was to detect the presence of E. gingivalis in Turkish healthy individuals and those with periodontal disease and to subtype the isolates using molecular techniques. Samples from the oral cavity of 94 individuals were taken and the presence of E. gingivalis was determined by PCR using primers for SsrRNA and the amplicons were then confirmed by DNA sequencing. Each participant completed a questionnaire that included demographic data, habits and lifestyle, as well as health status. The presence of E. gingivalis was detected in a total of 19 samples (11 patients and eight healthy individuals). Molecular characterization determined that 12 samples belonged to ST1 and seven samples belonged to ST2. The presence of E. gingivalis was higher in patients with periodontal disease than in healthy individuals, and this association was statistically significant (P < .05). This study constitutes the first report of molecular detection and subtyping of E. gingivalis in Turkey.


Introduction
T he oral ca vity has the second largest number and variety of micr oor ganisms in the body, housing millions of micr oor ganisms that form the or al micr obiota (Zhang et al. 2018 ).These micr oor ganisms form biofilms (dental plaque) and coexist in symbiotic harmony with the host, but when the microbial equilibrium is broken (dysbiosis) differ ent or al diseases such as dental caries and periodontal disease can occur (Dewhirst et al. 2010, Lourenço et al. 2014, Lamont et al. 2018, Yaseen et al. 2021, Martin-Garcia et al. 2022, Santos and Roldán 2023 ).Bacteria are the most abundant micr oor ganisms (For example: Streptococcus spp., Gemella spp ., Rothia spp ., Neisseria spp ., Haemophilus spp ., Prevotella spp., and Veillonela spp.) in oral microbiota in healthy individuals (Takeshita et al. 2016, Peters et al. 2017, Negrini et al. 2021 ).In addition to bacteria, the oral cavity can also harbour protozoa such as Entamoeba gingivalis and Trichomonas tenax , which received less attention in periodontal studies (Deo and Deshmukh 2019 ).Howe v er, ther e hav e been a some studies investigating the colonization of these parasites in the oral cavity of both healthy individuals and those with periodontal disease, yielding variable results (Bonner et al. 2014, Hassan et al. 2019, Trim et al. 2011, Yazar et al. 2016, Yaseen et al. 2021 ).
Entamoeba gingivalis is a protozoan microorganism that is mostly found in the human oral cavity and it has also been detected in the genitourinary tract (Bonner et al. 2014 ).Transmission occurs through contaminated food or oral equipment, mouth droplets, and kissing (Stensvold et al. 2021 ).Entamoeba gingivalis has a cosmopolitan distribution with a worldwide pr e v alence of 37% (Badri et al. 2021 ).Some studies proposed an association between E. gingivalis and periodontal diseases (Garcia et al. 2018b, Badri et al. 2021 ), and in a metagenomic study it w as sho wn that RNA le v els of E. gingivalis was ele v ated and the microbial diversity was reduced in the inflamed areas of the mouth (Deng et al. 2017, Bao et al. 2020, Badri et al. 2021 ).
To date, there are phylogenetically close two subtypes of E. gingivalis that have been identified as ST1 and ST2 (García et al. 2018a ).These subtypes are suggested to display different patterns of infectious behavior (Garcia et al. 2018b ).These subtypes share the same ecological niche in the oral ca vity, therefore , it is hard to separate their particular demographic , geographic , or clinical features with few studies done so far.
During the last few decades, many studies have been published on the investigation of the presence of E. gingivalis among healthy individuals and patients with se v er al dental diseases with variable results (Abualqomsaan et al. 2010, Ghabanchi et al. 2010, Özçelık et al. 2010, Trim et al. 2011, Bonner et al. 2014 ).Different or discr epant r esults may be r elated to the use of differ ent a ppr oac hes for the detection of E. gingivalis , the selection of different groups of patients, and the genetic diversity that, until now, is still somewhat unkno wn (Gar cía et al. 2018a , b ).In Turk e y ther e ar e not many studies on E. gingivalis and the genetic variation has to be explored (Abualqomsaan et al. 2010, Özçelık et al. 2010 , Yazar Table 1.Primers used in study.

Primer Sequence
Entam1 5 -GTTGA TCCTGCCAGT A TT A T A TG-3 Entam2 5 -CACT A TTGGAGCTGGAA TT AC-3 GEgFST1 5 -GA GA CGA TCCTGTTCT A TT AC-3 GEgFST2 5 GA GA C AATCCC A GTTGTTTGTA C3 EgST1/2-R 5 -ACT A TGT ACGTTCGTTCA TTCC-3 et al. 2016 ).The objective of this study was to detect the presence of E. gingivalis in both healthy individuals and patients with dental diseases by using molecular tec hniques.Additionall y, the study aimed to perform molecular c har acterization of the identified E. gingiv alis isolates, whic h had not pr e viousl y been done in Turk e y.

Study subjects and sample collection
Consecutive patients without any systemic disease ( n = 94), aged between 18 and 90 years, consulting in the dental clinic at Hacette pe Uni versity Be yte pe Hospital (Turk e y) were selected for this study.Ethical a ppr ov al for the study was granted by the ethics committee of Hacette pe Uni v ersity, Ankar a, Turk e y (GO 21/241).Sample collection was carried out between February 2021 and January 2022.From those who have not received antimicrobial therapy in the past 6 months were included in the study.The samples were taken from these patients' mouths with a sterile disposable cotton s wab.T he s w ab w as cir culated within the g ing iv al poc kets, mucosal surfaces, and tooth surface to increase the possibility of E. gingivalis detection.Each participant was asked to fill out a questionnaire that included demogr a phic, education, and income-r elated questions, as well as questions concerning knowledge about some lifestyle habits such as tap water usage, shared toothbrush usage, mouthwash usage, and smoking.In addition, in order to determine the general health status of the participants, the presence of c hr onic disease and regular drug usage history was also included in the questionnaire.

DN A extr action and amplifica tion
Initially, clinical samples in sterile saline were centrifuged at 1200 × g for 15 min in order to precipitate all the biological material.Genomic DN A w as extr acted fr om clinical samples using the DNA extraction Kit (GeneAll Biotechnology, Korea) according to the manufacturer's instructions .T he concentration and purity of all the obtained DN A w as measured by FLUOstar Omega Microplate Reader (BMG LABTECH) using LVis plate.In this study, to identify the subtypes as ST1 and ST2, a nested PCR was performed using the following primer sets Entam1/Entam2 and GEgFST1/GEgFST2-EgST1/2-R. The primer sequences used are given in Table 1 .For all PCR r eactions, pr e viousl y r eported conditions wer e a pplied for the time and temper atur e (Verweij et al. 2001, Garcia et al. 2018b ).Amplicons were visualized under ultraviolet light following electr ophor esis on 1.5% (w/v) a gar ose gel and products seen in the a ppr oximatel y 350 base-pair (bp) band size were considered positive.

Sequence data analyses
Obtained sequence c hr omatogr ams wer e examined using Finc hTV vie wer (Geospiza, Seattle, WA, USA) and determined the quality of generated nucleotide sequences.
Obtained sequences in this study that ar e pr esent in the NCBI database were compared using the BLAST algorithm ( http:// www.ncbi.nlm.nih.gov/BLAST/ ).Sequence data analysis was inter pr eted as pr e viousl y described (Boufana et al. 2014 ).In a few w or ds, data alignment w as performed in Mega version 7 and the phylogenetic tree was constructed using ClustalX (Larkin et al. 2007, Tam ur a et al. 2011 ).Additionall y, Ha pvie w pr ogr amme was used for generating haplotype networks (Salzburger et al. 2011 ).

Sta tistical anal ysis
Statistical analysis was performed using IBM SPSS Statistics Version 20 (SPSS Inc., Chicago, IL, USA).The chi-square test was used for group comparisons of categorical data.

Demographic and clinical characteristics of study groups
Of the 94 participants, 36 were classified as patients and the rest wer e consider ed healthy contr ols.Most of the participants wer e female (69.1%, 65/94).The age range of the participants was between 18 and 66 years, and the mean age was 30.Considering the educational status of the participants, most of them (73%, 69/94) were found to have a university and/or higher education degree.Most of the participants (72%, 68/94) were found to have middle/high income; some of them (13.8%,13/94) had c hr onic diseases .T he majority of the participants (69%, 65/94) indicated not using tobacco, while 87.2% (82/94) reported using spring water.Additionally, most of the participants (85.1%, 80/94) stated that they did not use mouthwash.

Detection and molecular char acteriza tion of E. gingivalis
A toal of 19 of the 94 samples (20.2%) were positive for E. gingivalis by nested PCR (with Entam1/Entam2 and GEgFST1-GEgFST2/EgST1/2); 11 of these 19 positive samples came from the oral cavity of patients with dental disease and the other eight came from healthy individuals .T he positivity r ates wer e found to be (11/36) in patients with dental disease and 13.8% (8/58) in healthy individuals.All sequences were confirmed as E. gingivalis by the BLAST algorithm.Regarding the subtypes, 12 samples were identified as ST1 and 7 were ST2.The phylogenetic tree was constructed using 22 different sequences (19 of the sequences are detected in this study) (Fig. 1 ).In addition, all the obtained sequences were submitted to GenBank (Some accession numbers of sequences: OP456213, OP456215, OP456304, OP422447, OQ932783O, and Q932784).Additionally, the analysis of haplotypes was conducted to visualize of genetic diversity among the obtained sequences mor e clearl y.The gener ated ha plotype network pr esents in Fig. 2 .

T he rela tionship betw een the presence of E. gingivalis and se ver al char acteristics of study groups
In this study, as an indicator of oral hygiene, tartar, periodontitis, smoking, tooth brushing habits, common tooth brush usa ge, mouthwash usa ge, drinking water pr efer ence, and pr esence of c hr onic diseases ar e the par ameters anal yzed.Clinical information and some lifestyle habits of E. gingiv alis positiv e patients wer e giv en in Table 2 .As a result, no significant relationships were found between the presence of E. gingivalis and patients' Figure 1.Maximum-likelihood phylogenetic analysis of small subunit ribosomal RNA partial sequences .T he obtained sequences in this study and some selected r efer ence sequences (AB282658 Entamoeba histolytica , D28490 E. gingivalis , KX027294 E. gingivalis ST2 kamaktli) were used to conduct a phylogenetic tree.
F igure 2. Haplotype netw ork generated using ssrRN A n ucleotide sequences of the E. gingivalis obtained in this stud y.
Table 2. Clinical information and some lifestyle habits of E. gingivalis positive patients.demogr a phics including age, gender, education, and income status ( P > .05),and no significant corr elations wer e observ ed between the presence of the parasite and the use of tap water, mouthwash, common toothbrushes, or tobacco ( P > .05).Howe v er, among the E. gingiv alis -positiv e individuals a significant association was observed between tobacco usage and dental diseases ( P = .012).In addition, the presence of E. gingivalis was significantly higher in patients with dental diseases compared to healthy individuals ( P = .02).Clinical information and some lifestyle habits of E. gingivalis positive healthy individuals wer e giv en in Table 3 .Any relationship was found between the presence of E. gingivalis and healthy individuals' demogr a phics suc h as a ge, gender, education, and income status ( P > .05).Additionall y, par asite pr esence and using tap w ater, mouthw ash, common tooth brush usage, and tobacco were not found associated ( P > .05).

Discussion
A total of 19 participants (19/94, 20.2%) were found positive for E. gingivalis by using molecular techniques.In detail, the positivity rates of E. gingivalis were found to be 30.5% (11/36) in patients with dental disease and 13.8% (8/58) in healthy individuals.There is no significant association between demogr a phics suc h as age, gender, educational status, income le v els, and some lifestyle habits such as using tap w ater, mouthw ash, and tobacco ( P > .05).Ho w e v er, Ar pa g and Kaya ( 2020 ) found a significant association between the presence of E. gingivalis and demographic data including gender, education status, frequency of dental visits, and brushing frequency (Arpa g and Kaya 2020 ).Additionally, another stud y re ported a higher pr e v alence of E. gingiv alis among individuals aged 45 years and above (Badri et al. 2021 ).Considering the a ge, the discr epancy in the r esults fr om differ ent r eports may be due to the difference in the average age of the participants.On the other hand, a significant correlation was found between the presence of E. gingivalis (30.5%, 11/36) and dental diseases ( P = .02)in this study.Our findings align with pr e vious studies that reported the detection of E. gingivalis in 29% and T. tenax in 2% of patients with g ing ivitis or periodontitis (Bardak et al. 1998 ).Similarly, another study investigating the density of E. gingivalis and T. tenax in microbial dental plaque found that 34.7% of the samples harbored E. gingivalis , while only 1.2% had T. tenax (Çeliksöz et al. 2001 ).
In the initial investigations conducted in the Eastern Anatolia ar ea, the pr e v alence of E. gingiv alis was determined to be 23.3% (Hakgüdener et al. 1998 ).Additionally, in another report involving 220 patients, the detection of E. gingivalis and/or T. tenax was found in 26.4% of the patients, with E. gingivalis identified in 21.8% of the cases and both E. gingivalis and T. tenax detected in 3.6% of the cases (Özçelık et al. 2010 ).In a study from the Aegean region, in 46 samples (33 with periodontal disease, 13 healthy subjects) in se v en (19.44%) samples E. gingiv alis was detected (Abualqomsaan et al. 2010 ).A pr e vious study fr om Centr al Anatolia has r eported that 60 (34.2%) out of 175 patients were found to be positive solel y for E. gingiv alis (Yazar et al. 2016 ).Ther e was no pr esence of par asite ar ound the healthy implants though out of 101 periimplantitis lesions, 31 (30.7%) of them was found E. gingivalis positive .T hese results support the consistency of our findings concerning the presence and prevalence of E. gingivalis in patients with dental diseases and healthy participants.
At least two molecular subtypes of E. gingiv alis hav e been described as ST1 and ST2 and both subtypes can be found in healthy people as commensal micr oor ganisms (Garcia et al. 2018b, Rahdar et al. 2019, Bao et al. 2020 ).Although E. gingivalis is considered , M: male, Y: yes, N: no, FS: first school, HS: high school, U: university, CD: cardiac disease, and HS: hypersensitivity.